Over the last 8 years, we identified and characterized patients with naturally occurring mutations in the orphan nuclear receptors, DAXl and SF1, and demonstrated that DAXl acts in part by inhibiting SF1- mediated transcription. Using targeted mutagenesis, we developed a murine Daxl knockout (KO) model and identified roles for Daxl in gonadal determination, testis development, and adult testis function. In this grant, we propose to extend these studies, which have provided unexpected insight into mechanisms of gonadal development. We propose 3 inter-related aims that focus on Daxl structure and function as a transcriptional represser, identify genetic pathways regulated by Daxl, and develop animal models to unravel the interplay of Daxl with other genes involved in testis development and function. Specifically, in Aim 1 we will identify molecular partners that mediate DAXl transcriptional repression. Naturally occurring DAX1 mutations will be identified and characterized to elucidate key structural domains required for DAX1 function in vivo. Candidate proteins identified in a yeast two-hybrid screen of an embryonic gonadal library will be further characterized. The goal of Aim 2 is to identify the genetic pathways regulated by Daxl using microarray analyses of genes expressed in wild type versus Daxl-deficient embryonic gonads at 12 dpc, a timepoint when Daxl expression diverges in males and females. Aim 3 is designed to explore the interaction of Daxl with other genetic pathways in vivo. Using mice that lack Daxl, we will explore the gonadal phenotypes of mice with selective rescue of Daxl in various cell types. In addition, Daxl-deficient mice will be crossed to other strains with alterations in genetic pathways proposed to intersect with Daxl (e.g., Sfl, Sry, Ptc). Success in these aims should provide a critical link between DAX1 and transcriptional repression pathways that link a variety of other transcription factors involved in gonadal development.